The epidermal growth factor receptor (EGFR) is often described as a ?prototypic? receptor tyrosine kinase (RTK). As one of the first single transmembrane domain receptors for which ligand-induced dimerization was reported, the ?traditional? view of EGFR (and other RTKs) has been of a binary ?off?/?on? switch. Our previous work, together with recent work on similar receptors, instead argues that signaling by receptors of this type is much more ?graded? ? and that EGFR and other receptors can discriminate between different ligands. Indeed, we showed that different EGFR-activating ligands can induce receptor dimers with distinct structures ? leading to different signaling kinetics and orthogonal cellular outcomes. Remarkably, the key residues that define the differences between the structures of these dimers coincide with the hotspots for extracellular EGFR mutations in glioblastoma. We therefore hypothesize here that EGFR mutations in GBM may exert at least part of their effect by altering the nature of the signaling response to different EGFR ligands. This would open the possibility of ?correcting? EGFR signaling in cancer if antibodies or other agents could be developed to reprogram them as ?allosteric microprocessors? by analogy with biased agonists for G-protein coupled receptors. Primary resistance to EGFR inhibitors in glioblastoma and in lung cancer is an important clinical problem that limits success with existing EGFR-targeted tyrosine kinase inhibitors (TKIs). Static structural views of kinase domains have not allowed satisfying explanations for the relative abilities of TKIs to inhibit different variants ? leaving the origins of primary resistance and of mutant selectivity (e.g. of osimertinib) unclear and difficult to solve. New instances of acquired TKI resistance suffer from the same problem. Our studies using hydrogen-deuterium exchange mass spectrometry (HDX-MS) alongside enzymology suggest that the key may lie in the effects of both the mutations and the TKIs themselves on structural dynamics ? particularly in the case of 3rd generation covalent EGFR inhibitors that associate with their targets in multiple steps. With this background, the key overall goal of this proposal is to identify specific behavior of EGFR variants seen in cancer that can explain their altered signaling properties and altered sensitivity to inhibitors. We apply a range of biophysical, structural, biochemical, and cellular approaches to interrogate signaling at several levels of resolution. In addition to answering key mechanistic questions for EGFR, our results should illuminate potential new avenues for therapeutic intervention. Our Specific Aims ask the following questions: 1 How do disease-associated extracellular mutations in EGFR family members affect signaling specificity and kinetics? 2 Can structural dynamics explain primary kinase inhibitor resistance of exon 19 EGFR variants in lung cancer, and selectivity of 3rd generation covalent inhibitors?